Silk Proteins as Biomaterial Additives for DMSO-Reduced Cryopreservation

TL;DR

This paper explores using silk proteins to reduce DMSO in cell freezing, improving cell survival and function after thawing.

Contribution

The study introduces silk fibroin and sericin as effective additives to reduce DMSO toxicity in cryopreservation.

Findings

Silk proteins alone cannot replace DMSO for cell survival during freezing.

Hybrid formulations with silk and 5% DMSO improved cell viability and recovery.

Silk-based cryopreservation supports better cytoskeletal integrity post-thaw.

Abstract

Background: Cryopreservation is a key enabling technology for cell-based therapies and regenerative medicine; however, the toxicity associated with permeating cryoprotective agents such as dimethyl sulfoxide (DMSO) remains a major limitation, particularly for applications requiring repeated cell administration or long-term storage. Methods: In this study, silk-derived proteins, namely silk fibroin and silk sericin, were investigated as biomaterial-based cryoprotective additives to enable DMSO-sparing cryopreservation strategies. Mouse fibroblasts (3T3) were cryopreserved at −80 °C using conventional DMSO-based media, silk-only formulations, and hybrid formulations combining silk proteins with reduced DMSO concentrations. Post-thaw cell adhesion, metabolic activity, membrane integrity, and cytoskeletal organization were systematically evaluated over a 7-day culture period. Results: Complete replacement of DMSO with silk proteins was insufficient to ensure cell survival, confirming the essential role of permeating cryoprotectants for intracellular protection. In contrast, formulations combining silk fibroin or sericin with 5% (v/v) DMSO supported robust post-thaw viability, preserved cytoskeletal architecture, and promoted favorable recovery kinetics, with cell viability consistently exceeding established biocompatibility thresholds and higher than samples with DMSO alone. Conclusions: These findings support the integration of biomaterial-based components into hybrid cryopreservation formulations and provide design principles relevant to the preservation of more complex multicellular systems.

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Full text

1. Introduction

Cryopreservation is a cornerstone technology in cell-based therapies, tissue engineering, and regenerative medicine, as it enables long-term storage, centralized manufacturing, and on-demand distribution of living products [1]. While current protocols are relatively well established for many suspension cell types, the development of robust cryopreservation strategies for complex three-dimensional constructs, tissues, and ultimately whole organs remains a major bottleneck for translation [1,2,3]. Recent reviews emphasize that successful organ banking will depend on protocols that first achieve reliable, high-quality preservation at the cellular level and then scale to multicellular architectures without compromising viability, function, or structural integrity [1].

During freezing and thawing, cells are exposed to multiple injurious stresses, including extracellular ice formation, solute concentration, osmotic and mechanical stress during dehydration and rehydration, and, in some settings, oxidative damage [2,4]. In tissues and organs, these stresses are compounded by heterogeneous heat and mass transfer, leading to local differences in cooling/warming rates, ice nucleation, and cryoprotectant distribution [2]. To mitigate cryoinjury, permeating and non-permeating cryoprotective agents (CPA) are routinely combined. Permeating agents such as dimethyl sulfoxide (DMSO) or ethylene glycol diffuse across the plasma membrane and reduce intracellular ice formation by lowering intracellular water activity and moderating osmotic gradients, while non-permeating solutes (e.g., sugars, polymers, hydroxyethyl starch) promote controlled cell dehydration and contribute to membrane and protein stabilization [4,5,6,7].

Among permeating agents, DMSO has been the “gold standard” for most mammalian cell systems since the early days of cryobiology and is still widely used at concentrations around 10% (v/v) in clinical cell products [5,8].

However, accumulating evidence shows that DMSO is far from biologically inert. In vitro, DMSO can increase reactive oxygen species, apoptosis and plasma membrane pore formation at concentration higher than 10 v/v%, with potential consequences for cell differentiation and function [9,10,11,12].

Clinically, infusion of DMSO-containing cell products has been associated with acute adverse reactions such as nausea, vomiting, cardiovascular events, and rare cases of neurotoxicity, prompting efforts to limit the total DMSO dose delivered to patients [13,14]. These concerns, together with the need for time-consuming washout steps after thawing, have driven a broad effort to develop DMSO-free or DMSO-sparing formulations that maintain post-thaw cell quality while reducing toxicity [15,16].

For many clinically relevant cell types, DMSO remains necessary to achieve acceptable survival, and there is a growing interest in strategies that reduce, rather than eliminate, DMSO by combining it with different protective components [17,18].

In parallel, some biomaterials are emerging as promising cryoprotective matrices that can modulate ice formation and mechanical stresses during freezing and thawing. Natural polymers such as hyaluronic acid, alginate, chitosan, and protein-based scaffolds, including silk fibroin and sericin, are being explored to preserve three-dimensional constructs and bioprinted tissues while supporting structural integrity and post-thaw function [19,20].

Silk proteins are particularly attractive because of their established biocompatibility, tunable secondary structure, and amphiphilic domains that strongly interact with water [21,22,23,24]. Along with the well-known biological properties of silk proteins, including bioactivity and tissue regeneration, recent advances have highlighted the potential of silk-derived proteins, including both fibroin and sericin, as cryoprotective additives. These proteins can modulate ice nucleation and recrystallization, stabilize cellular membranes, and improve post-thaw recovery across different stem-cell types.

Recent work has shown that silk fibroin, used as a CPA additive for human bone-derived mesenchymal stem cells, can suppress both ice formation during cooling and devitrification-induced recrystallization during warming [25,26].

Despite these advances, systematic evaluation of silk proteins as cryoprotective components is not fully understood. The relative contributions of DMSO and silk proteins require more investigations, by directly comparing conditions that use DMSO alone, silk alone, or combinations in which DMSO is partially substituted by silk, while quantifying not only immediate post-thaw viability but also subsequent cell attachment, proliferation, and functional recovery. Such mechanistic insights at the cell level are essential if silk-based CPAs are to be rationally integrated into cryopreservation strategies for more complex tissue constructs and for organ-scale preservation where CPA toxicity and multi-scale ice damage are key obstacles.

On this basis, the present study investigates the cryopreservation of fibroblasts in media containing DMSO as the sole CPA, silk proteins alone, and reduced concentrations of DMSO supplemented with silk proteins. In addition, the experiments were conducted at −80 °C, and not in liquid nitrogen (−196 °C) because cells that remain viable under these conditions are expected to maintain viability at even lower temperatures. By directly comparing these conditions, this study aims to clarify whether silk proteins can partially substitute for DMSO, thereby enabling lower DMSO exposure while preserving or even enhancing post-thaw cell viability and recovery.

2. Materials and Methods

2.1. Cell Freezing Conditions

3T3 fibroblasts were frozen simultaneously at −80 °C in different freezing media: (i) 3T3 frozen in traditional medium, such as 90% FBS (Fetal Bovine Serum) and 10% DMSO (Dimethyl sulfoxide) (CRL); (ii) 3T3 frozen in 90%FBS, 5% Silk Fibroin solution (5% w/v) and 5% DMSO (s-SF + DMSO); (iii) 3T3 frozen in 90%FBS and 10% Silk Fibroin solution (5% w/v) (s-SF − DMSO); (iv) 3T3 frozen in 90%FBS, 5% Silk Fibroin solution from powder (5% w/v) and 5%DMSO (p-SF + DMSO); (v) 3T3 frozen in 90%FBS and 10% Silk Fibroin solution from powder (5% w/v) (p-SF − DMSO); (vi) 3T3 frozen in 90%FBS, 5% Silk Sericin solution from powder (5% w/v) and 5%DMSO (p-SS + DMSO); (vii) 3T3 frozen in 90%FBS and 10% Silk Sericin solution from powder (5% w/v) (p-SS − DMSO). Cells in the freezing media were placed directly at −80 °C; two biological replicates were prepared for each media. Silk fibroin and sericin solutions derived from powder (p-SF and p-SS) were prepared by dissolving them in water at a concentration of 5% w/v, corresponding to that of the silk fibroin solution (s-SF) (5% w/v) that was extracted directly in aqueous solution. All solutions were sterilized in autoclave at 121 °C for 15 min prior to their use in the preparation of the freezing media. Silk fibroin solution, silk fibroin powder and silk sericin powder were kindly provided by Caresilk S.r.l.s. (Lecce, Puglia, Italy), whereas DMSO was purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Cell Viability Evaluation

3T3 fibroblast frozen at −80 °C in the different media were thawed after 1 month in the thermostatic bath at 37 °C and immediately placed in cell culture flasks. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented with 10% FBS, 1% antibiotic solution (100 U/mL penicillin and 100 µg/mL streptomycin), and 2 mM L-glutamine. Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO_2_ (Heracell incubator, Thermo Scientific, Waltham, MA, USA), and the culture medium was refreshed every two days. Cells were observed under a light microscope after 48 h, and cell adhesion was evaluated prior to performing the cell viability assays. Subsequently, 10 × 10^4^ viable cells were transferred into 24-well plates, and the experimental time course for the assessment of cell viability parameters was initiated.

2.3. MTT Assay

The metabolic activity of 3T3 fibroblasts, previously frozen in the different media (CRL, s-SF + DMSO, s-SF − DMSO, p-SF + DMSO, p-SF − DMSO, p-SS + DMSO, p-SS − DMSO) and cultured in supplemented DMEM media, was determined using the MTT colorimetric assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma-Aldrich). The cell viability analysis was carried out in triplicate after 3, 5, and 7 days of incubation. Briefly, MTT stock solution (5 mg/mL in PBS) was diluted in fresh culture medium to a final concentration of 0.5 mg/mL and added to each well. After 4 h of incubation at 37 °C, the resulting purple formazan crystals were solubilized in DMSO. Absorbance values were then recorded at 540 nm using a Multimode Plate Reader EnVision (PerkinElmer, Waltham, MA, USA) [27].

2.4. Live/Dead Assay

Cell viability and membrane integrity of 3T3 fibroblasts, previously frozen in the different media (CRL, s-SF + DMSO, s-SF − DMSO, p-SF + DMSO, p-SF − DMSO, p-SS + DMSO, p-SS − DMSO), were further investigated through a Live/Dead fluorescence assay. The analysis was performed after 5 and 7 days of culture. Cells grown directly on coverslips were incubated at 37 °C for 15 min with a staining solution containing 2 µmol/L calcein-AM (acetoxymethoxy) and 2 µmol/L propidium iodide in PBS. Subsequently, live (green) and dead (red) cells were visualized using a fluorescence microscope (Axio Vert A1, Zeiss, Oberkochen, Germany) equipped with 20× magnification optics. Images were processed and analyzed using AxioVision software (Zeiss ZEN 3.11) [28].

2.5. Cytoskeleton Architecture Analysis

The organization of the cytoskeleton actin filaments in 3T3 fibroblasts previously frozen in the different media (CRL, s-SF + DMSO, s-SF − DMSO, p-SF + DMSO, p-SF − DMSO, p-SS + DMSO, p-SS − DMSO) was examined after 5 and 7 days of incubation. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by permeabilization with 0.5% Triton X-100 for 10 min and rinsing with PBS. Actin filaments were labeled with tetramethylrhodamine isothiocyanate (TRITC) - conjugated phalloidin (Sigma-Aldrich) prepared in PBS at a concentration of 1 µL/mL, while nuclei were counterstained with 0.5 mg/mL DAPI (Invitrogen, Waltham, MA, USA). Fluorescence imaging was performed at 40× magnification using an Axio Vert A1 microscope (Zeiss), and cytoskeletal organization was analyzed with Zeiss ZEN 3.11 software [28].

2.6. Statistical Analyses

Statistical evaluations were performed using GraphPad Prism version 8.0.1. Data from the MTT assay were analyzed through two-way ANOVA followed by multiple comparison tests. A p-value < 0.05 was considered as indicative of statistical significance [29].

3. Results

3.1. Cell Adhesion Assessment Results

As described in the previous section, 3T3 fibroblasts were cryopreserved using the conventional freezing medium (CRL) and several experimental formulations containing silk-derived proteins, namely s-SF + DMSO, s-SF − DMSO, p-SF + DMSO, p-SF − DMSO, p-SS + DMSO, and p-SS − DMSO. After one month of storage at −80 °C, the cells were thawed and cultured under standard conditions. Two days post-thawing, cell adhesion and viability were assessed by optical microscopy. As shown in Figure 1, 3T3 fibroblasts frozen in the conventional medium (CRL) remained viable, as well as those preserved in the experimental cryoprotective solutions where silk proteins replaced 50% of DMSO (s-SF + DMSO, p-SF + DMSO, p-SS + DMSO). In contrast, cells frozen in formulations in which DMSO was completely replaced by silk protein solutions (s-SF − DMSO, p-SF − DMSO, p-SS − DMSO) did not survive (Figure 1).

Viable 3T3 fibroblasts were subsequently cultured for a 7-day time course, with adhesion evaluated at days 3, 5, and 7. Optical microscopy images revealed adherent 3T3 fibroblasts displaying the typical spindle-shaped morphology and progressive proliferation over the experimental period (Figure 2).

3.2. MTT Assay Results

The MTT assay was performed to evaluate the impact of the different freezing media (CRL, s-SF + DMSO, p-SF + DMSO, p-SS + DMSO) on the viability of 3T3 fibroblasts. In the present study, increased absorbance was interpreted as an indicator of enhanced cell viability, since metabolically active cells reduce the MTT reagent to insoluble purple formazan crystals. A significant increase in absorbance at 540 nm was observed from day 3 to day 7 in the p-SF and s-SF experimental groups, as well as in the p-SS group. In s-SF experimental group the absorbance significantly increased from day 3 to day 5. In contrast, the CRL group showed no significant changes in absorbance across the different time points (CRL: day 3 vs. day 5, p > 0.05; day 3 vs. day 7, p > 0.05; day 5 vs. day 7, p > 0.05. s-SF: day 3 vs. day 5, p < 0.05; day 3 vs. day 7, p > 0.05; p > 0.05; day 5 vs. day 7, p > 0.05. p-SF: day 3 vs. day 5, p > 0.05; day 3 vs. day 7, p < 0.05, day 5 vs. day 7, p > 0.05. p-SS: day 3 vs. day 5, p > 0.05; day 3 vs. day 7, p < 0.05, day 5 vs. day 7, p > 0.05).

Moreover, the absorbance values of all experimental groups were significantly higher than those of the CRL group on day 5 (CRL vs. s-SF, CRL vs. p-SF, CRL vs. p-SS, p < 0.05) and this difference remained significant for the s-SF and p-SF groups on day 7 (CRL vs. s-SF, CRL vs. p-SF, p < 0.05) (Figure 3A). These findings demonstrate that the different freezing media tested herein do not compromise cell viability after the thawing, showing good viability after 7 days of cultures. To further quantify these results, cell viability was expressed as a percentage relative to the control at each time interval. The results obtained for all experimental groups showed cell viability values above 70% at all time points, indicating that the cells remained viable up to day 7, according with ISO 10993-5:2009 [30] which defines a reduction greater than 30% as indicative of cytotoxicity [31]. Statistical analysis revealed a significant difference in s-SF group from day 3 to day 5 (p < 0.05), as well as in p-SF from day 3 to day 7 (p < 0.05) (Figure 3B).

3.3. Live and Dead Assay Results

The cells viability of 3T3 fibroblasts frozen in different conditions CRL, s-SF + DMSO, p-SF + DMSO, p-SS + DMSO) was assessed by means of a Live/Dead fluorescence assay performed on days 5 and 7. Live and dead cells were identified by green and red fluorescence, respectively. Fluorescence imaging at both time points revealed widespread survival in all experimental groups, comparable to the control (Figure 4). The absence of red fluorescence confirmed the non-cytotoxic nature of the novel condition media containing silk proteins.

3.4. Cytoskeleton Architecture Structure

The cytoskeletal arrangement of 3T3 fibroblasts frozen in different conditions CRL, s-SF + DMSO, p-SF + DMSO, p-SS + DMSO) was examined through phalloidin–TRITC staining on days 5 (Figure 5A) and 7 (Figure 5B). Cells frozen in media containing silk proteins such as (i) s-SF + DMSO, (ii) p-SF + DMSO, (iii) p-SS + DMSO, displayed a well-organized actin network comparable to that observed in the control group at each time point. Microscopic images acquired at 40× magnification (Figure 5) showed intact and regularly distributed actin filaments, confirming that the novel cell freezing media does not affect the cytoskeletal organization up to day 7, thus supporting cell compatibility.

4. Discussion

The present study systematically evaluated the role of silk-derived proteins, namely silk fibroin and silk sericin, as cryoprotective additives in fibroblast cryopreservation, with the specific aim of assessing whether these biomaterials can partially substitute for dimethyl sulfoxide (DMSO) while preserving post-thaw cell viability and functional recovery. By directly comparing conventional DMSO-based formulations, silk-only conditions, and DMSO-reduced formulations supplemented with silk proteins, this work provides experimental insight into the relative and combined contributions of biomaterial-based cryoprotectants. In the present study, experiments were conducted at −80 °C, as this temperature represents a stringent condition for assessing cell survival. Cells that remain viable under these conditions are expected to maintain viability at even lower temperatures, such as during storage in liquid nitrogen.

The initial adhesion assessment clearly demonstrated that complete replacement of DMSO with silk proteins, irrespective of whether fibroin or sericin was used, was insufficient to ensure cell survival following freezing and thawing. Indeed, cells cryopreserved in DMSO-free silk-containing formulations failed to adhere and proliferate after thawing, indicating extensive cryoinjury. This finding is consistent with the established role of permeating cryoprotectants in suppressing intracellular ice formation and buffering osmotic stress during cooling and rewarming, functions that cannot be fully compensated by non-permeating or macromolecular additives alone [4,5]. Similar observations have been reported for other non-permeating cryoprotectants, including sugars and polymers, which typically require combination with permeating agents to achieve adequate intracellular protection [6,7]. In contrast, all formulations in which silk proteins were combined with a reduced concentration of DMSO (5% v/v) supported robust post-thaw adhesion, proliferation, and metabolic activity, comparable to or exceeding those observed with the conventional 10% DMSO formulation, particularly in the case of fibroin.

Quantitative MTT analysis further revealed that cells cryopreserved in DMSO-reduced media exhibited a progressive increase in metabolic activity over the 7-day culture period, whereas cells frozen in the conventional DMSO formulation showed relatively stable absorbance values over time. These data suggest that silk-containing formulations not only preserve viability but may also promote more favorable post-thaw recovery kinetics. Importantly, cell viability remained consistently above the 70% threshold, confirming the absence of cytotoxic effects associated with the silk-based additives and supporting their biocompatibility [20,30].

Live/Dead staining and cytoskeletal analysis corroborated these findings at the morphological level. Cells cryopreserved with silk proteins and reduced DMSO displayed intact plasma membrane integrity and well-organized actin cytoskeletons comparable to those observed in control cells. Preservation of cytoskeletal architecture is a critical indicator of functional recovery, as cytoskeletal disruption has been linked to impaired adhesion, migration, and mechanotransduction following cryoinjury [2,4]. From a mechanistic perspective, the results align with recent reports demonstrating that silk fibroin can suppress ice nucleation during cooling and inhibit devitrification-induced ice recrystallization during warming, thereby reducing both primary and secondary ice-related damage [25,26]. In human bone-derived mesenchymal stem cells, the addition of silk fibroin enabled post-thaw viabilities comparable to those achieved using commercial macromolecular cryoprotectants, while preserving key cellular functions [25]. Regulation of ice crystals formation during cooling process observed in presence of fibroin was attributed to its strong hydration capability compared with the cryopreservation medium alone [25]. Hydrogen bonding plays a central role in the behavior of silk fibroin in aqueous environments. The repetitive primary sequence of silk fibroin, rich in glycine and serine residues, favors the formation of intermolecular hydrogen bonds that stabilize β-sheet domains, and these polar side chains are also capable of interacting with surrounding water molecules. NMR studies have shown distinct populations of water bound to silk fibroin, consistent with hydrogen-mediated interactions between water and protein domains. Additionally, functional studies indicate that serine side chains readily participate in hydrogen bonding, influencing adhesive and interfacial properties of silk proteins [32,33,34]. Although the present study used robust 3T3 fibroblasts as a proof-of-concept, there is emerging evidence that silk fibroin can also be applied to more clinically relevant and cryosensitive cell types. For example, silk fibroin has been successfully employed as a cryoprotective agent for human bone marrow-derived mesenchymal stem cells, with the addition of fibroin regulating ice formation and preserving post-thaw viability and function [25].

Importantly, the inability of silk proteins alone to replace DMSO highlights the continued necessity of permeating cryoprotectants for effective intracellular protection, particularly under conventional slow-freezing conditions. Rather than supporting a fully DMSO-free approach, the present data reinforce the emerging consensus that hybrid strategies combining reduced concentrations of permeating CPAs with biocompatible macromolecular additives represent a more realistic and immediately translatable pathway toward safer cryopreservation protocols.

Taken together, this study provides experimental evidence that silk fibroin and sericin can function as effective DMSO-sparing cryoprotective additives, enabling a significant reduction in DMSO concentration without compromising post-thaw viability, morphology, or cytoskeletal integrity.

Such strategies may be particularly relevant for the cryopreservation of complex multicellular systems, including engineered tissues and bioprinted constructs, where both cellular viability and structural integrity must be preserved across multiple length scales.

5. Conclusions

This study evaluated silk fibroin and silk sericin as biomaterial-based cryoprotective additives in the cryopreservation of fibroblasts, with the aim of reducing exposure to dimethyl sulfoxide while maintaining post-thaw viability and functional recovery. Through a direct comparison of conventional DMSO-based formulations, silk-only conditions, and DMSO-reduced media supplemented with silk proteins, the relative contributions and limitations of silk-derived cryoprotectants were systematically assessed. The results demonstrated that silk proteins alone are insufficient to replace DMSO under the freezing conditions investigated, underscoring the continued necessity of permeating cryoprotective agents for effective intracellular protection. However, when combined with a reduced DMSO concentration (5% v/v), both silk fibroin and sericin supported robust post-thaw adhesion, metabolic activity, membrane integrity, and cytoskeletal organization over extended culture periods, with outcomes that were comparable to, and in some cases even higher for fibroin, than those observed with the standard formulation.

The present findings support a model in which silk-derived proteins act as complementary cryoprotective components and contribute to extracellular ice control and improved post-thaw recovery, enabling meaningful reductions in DMSO concentration without compromising cell quality.

Such strategies may be particularly advantageous for applications involving sensitive cell types, repeated dosing, or the cryopreservation of complex multicellular systems, including engineered tissues and three-dimensional constructs.